in vitro wound healing assay

HMEC cells were split on 100 mm dishes and plated to reach high confluency. Cells then were starved in MCDB medium (IITD PAN, Wroclaw, Poland) with 0.1% of FBS for 12 h. Next step was to treat 2 dishes with 25 μM of C01L_F03 and 2 dishes with 25 μM of STX-0119. After 12 h of pre-incubation with C01L_F03 or STX-0119 scratches in these dishes were made. Another set of 2 dishes was treated with 10 μM of STATTIC 12 h before pictures were taken. At the same time 10 ng/ml of IFNγ and 1 μg/ml of LPS were added to one dish from each pair treated with C01L_F03, STX-0119 or STATTIC. Additionally, scratches were also made in set of 2 dishes that remained not treated with any inhibitor, one was used as an untreated control and to the second only IFNγ and LPS were added. Pictures were taken with Axio Observer.Z1 Microscope (Zeiss) after 12 h since the moment when scratches were made. The images acquired for each sample from two independent repeats were further analyzed quantitatively by ImageJ (36). For each image, 20 distances between one side of scratch and the other were measured at certain intervals (μm). By comparing the images from or inhibitor (with or without IFNγ+LPS treatment) to control, the distances of each scratch closure were obtained. Analysis of HMECs migration according to wound healing in vitro, which was performed according to Liang et al. (37).